As a topoisomerase I inhibitor, the prodrug irinotecan has been widely used in the chemotherapy of metastatic cancers. However, the transformation from irinotecan to its active metabolite SN-38 is slow and non-selective in cancer cells, which is not desired for clinical oncotherapy. Additionally, the fluorescence turn-on response of irinotecan is poor, prohibiting real-time observations on SN-38 release. Here, we developed a new prodrug (BDD-SN38) that is well suited for real-time monitoring SN-38 release and reducing its activation in normal cells. The fluorescence and activity of BDD-SN38 was significantly quenched by dinitrobenzene group. The in vitro and in cell results showed that BDD-SN38 could be selectively and rapidly activated by hydrogen sulfide (H2S) to obtain significantly enhanced fluorescence and activity. Moreover, BDD-SN38 achieved remarkable toxicity towards H2S-overexpressed cancer cells. Overall, the H2S-responsive prodrug developed here was proved to be a promising candidate for real-time monitoring SN-38 release and improving its therapeutic effect.